Laura Fernández-Sirera

Evaluation of the efficacy of commercial vaccines against bluetongue virus serotypes 1 and 8 in experimentally infected red deer (Cervus elaphus)

Red deer (Cervus elaphus) is a widespread and abundant species susceptible to bluetongue virus (BTV) infection. Inclusion of red deer vaccination among BTV control measures should be considered. Four out of twelve BTV antibody negative deer were vaccinated against serotype 1 (BTV-1), and four against serotype 8 (BTV-8). The remaining four deer acted as unvaccinated controls. Forty-two days after vaccination (dpv), all deer were inoculated with a low cell passage of the corresponding BTV strains. Serological and virological responses were analyzed from vaccination until 28 days after inoculation (dpi). The vaccinated deer reached statistically significant (P<0.05) higher specific antibody levels than the non vaccinated deer from 34 (BTV-8) and 42 (BTV-1) dpv, maintaining stable neutralizing antibodies until 28 dpi. The non vaccinated deer remained seronegative until challenge, showing neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of the non vaccinated deer from 2 to 28 dpi, whereas no BTV RNA was found in the vaccinated deer. BTV was isolated from the blood of non vaccinated deer from 7 to 28 dpi (BTV-1) and from 9 to 11 dpi (BTV-8). BTV RNA could be identified by RT-PCR at 28 dpi in spleen and lymph nodes, but BTV could not be isolated from these samples. BT-compatible clinical signs were inapparent and no gross lesions were found at necropsy. The results obtained in the present study confirm that monovalent BTV-1 and BTV-8 vaccines are safe and effective to prevent BTV infection in red deer. This finding indicates that vaccination programs on farmed or translocated red deer could be a useful tool to control BTV.

Experimental infection with chamois border disease virus causes long-lasting viraemia and disease in Pyrenean chamois (Rupicapra pyrenaica)

Since 2001, severe outbreaks of disease associated with border disease virus (BDV) infection have been reported in Pyrenean chamois. The disease is characterized by variable degrees of cachexia, alopecia and neurological manifestations prior to death. The aim of this study was to investigate this disease under experimental conditions. To assess viral virulence, humoral immune response, dissemination and probable routes of transmission, seven chamois (five seronegative and two seropositive for BDV) were inoculated with a BDV isolated from a naturally infected chamois. A group of three chamois were maintained as uninfected controls. The five seronegative chamois became viraemic from day 2 post-inoculation (p.i.) until their death (three animals) or the end of the experiment (on day 34 p.i.) and developed neutralizing antibodies from day 18 p.i. until the end of the study. Continuous shedding of the virus was detected by RT-PCR in oral, nasal and rectal swabs in viraemic chamois from day 5 p.i. Despite none of the viraemic chamois showing obvious neurological signs, all of them had a non-suppurative meningoencephalitis as seen in naturally infected chamois. The two inoculated BDV-seropositive chamois did not become viraemic. This study confirms that BDV is the primary agent of the disease that has been affecting chamois populations in recent years in the Pyrenees and that previously acquired humoral immunity is protective.

Two Different Epidemiological Scenarios of Border Disease in the Populations of Pyrenean chamois (Rupicapra p. pyrenaica) after the First Disease Outbreaks

Since 2001 several outbreaks of a new disease associated with Border disease virus (BDV) infection have caused important declines in Pyrenean chamois (Rupicapra pyrenaica) populations in the Pyrenees. The goal of this study was to analyze the post-outbreak BDV epidemiology in the first two areas affected by disease with the aim to establish if the infection has become endemic. We also investigated if BDV infected wild and domestic ruminants sharing habitat with chamois. Unexpectedly, we found different epidemiological scenarios in each population. Since the disease outbreaks, some chamois populations recuperated quickly, while others did not recover as expected. In chamois from the first areas, prevalence was high (73.47%) and constant throughout the whole study period and did not differ between chamois born before and after the BDV outbreak; in all, BDV was detected by RT-PCR in six chamois. In the other areas, prevalence was lower (52.79%) and decreased during the study period; as well, prevalence was significantly lower in chamois born after the disease outbreak. No BDV were detected in this population. A comparative virus neutralisation test performed with four BDV strains and one Bovine viral diarrhoea virus (BVDV) strain showed that all the chamois had BDV-specific antibodies. Pestivirus antibodies were detected in all the rest of analyzed species, with low prevalence values in wild ruminants and moderate values in domestic ruminants. No viruses were detected in these species. These results confirm the hypothesis that outbreaks of BDV infection only affect the Pyrenean chamois, although other wild ruminants can occasionally be infected. In conclusion, two different scenarios have appeared since the first border disease outbreaks in Pyrenean chamois: on the one hand frequent BDV circulation with possible negative impact on population dynamics in some areas and on the other, lack of virus circulation and quick recovery of the chamois population.

Border Disease Virus: An Exceptional Driver of Chamois Populations Among Other Threats

Though it is accepted that emerging infectious diseases are a threat to planet biodiversity, little information exists about their role as drivers of species extinction. Populations are also affected by natural catastrophes and other pathogens, making it difficult to estimate the particular impact of emerging infectious diseases. Border disease virus genogroup 4 (BDV-4) caused a previously unreported decrease in populations of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Spain. Using a population viability analysis, we compared probabilities of extinction of a virtual chamois population affected by winter conditions, density dependence, keratoconjunctivitis, sarcoptic mange, and BD outbreaks. BD-affected populations showed double risk of becoming extinct in 50 years, confirming the exceptional ability of this virus to drive chamois populations.

Surveillance of border disease in wild ungulates and an outbreak in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Andorra.

The Principality of Andorra is surrounded by areas in which Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations were severely affected by infection with border disease virus (BDV) which caused disease outbreaks between 2001 and 2009. Nevertheless, the Andorran chamois populations were not affected during this period. In light of the severe impact of BDV on several of the neighboring Pyrenean chamois populations, we monitored local Andorran populations in an effort to detect pestivirus antibodies and BDV in wild ungulates. In addition, an episode of mortality between 2009 and 2010 in chamois was investigated. We analyzed samples (spleen or serum) from 175 Pyrenean chamois, 284 European mouflon (Ovis orientalis musimon), 13 roe deer (Capreolus capreolus capreolus), and five wild boars (Sus scrofa castilianus). With the exception of three dead chamois found between 2009 and 2010, all samples came from healthy animals hunted during the hunting season. A commercial blocking enzyme-linked immunosorbent assay (ELISA) was used to test sera for antibodies against pestivirus. Positive sera were tested with a comparative virus neutralization test (VNT) using three BDV strains and a bovine viral diarrhea virus strain. Reverse-transcription–polymerase chain reaction (RT-PCR) was performed on all sera and spleen homogenates. Antibodies against pestivirus were detected by ELISA in four of the 69 chamois (5%; 95% CI= 1.29–13.11). The VNT confirmed three of these chamois were infected with a BDV. Viral RNA was detected by RT-PCR in three chamois—one apparently healthy animal hunted in 2009 and two dead animals. Viral sequences showed that the three chamois were infected with a BDV-4, the same genotype that was involved in previous episodes of mortality in the Pyrenees. Although Pyrenean chamois from Andorra had had little contact with the pestiviruses until 2009, in this year BDV was associated with a severe disease outbreak.

Survey of Pestivirus infection in wild and domestic ungulates from south-western Italian Alps

The transmission of pestiviruses between domestic and wild ruminants has not been documented in communal alpine pastures shared between wildlife and livestock. The aim of this study was to investigate the role of domestic and wild ungulates species from Varaita Valley (SW Italian Alps) in the epidemiology of Pestivirus infections. Sera from free-ranging alpine chamois (Rupicapra rupicapra) and roe deer (Capreolus capreolus) were collected from 1994 to 2009 and 2001 to 2009, respectively. Also, sera from cattle and sheep sampled in 2009 were studied. Sera were tested for the presence of antibodies against pestivirus with an ELISA assay. Sera from positive animals were subsequently tested with a comparative virus neutralisation test using the BVDV-NADL and BDV-137/4 strains. Sera were tested for the presence of pestiviral antigen and the presence of viral RNA with a commercial ELISA assay and RT-PCR. Antibodies against Pestivirus were detected in 132 out of 312 (42%) chamois, in 30 out of 175 (17%) cattle and 6 out of 24 (25%) sheep. No antibodies were found in roe deer. No Pestivirus antigen or RNA was detected in any of the samples. Results indicate circulation of pestiviruses among the studied chamois, cattle and sheep populations. However the role of wild ungulates in the dynamics of Pestivirus infection is still unknown and monitoring the presence of these viruses in wild ungulates would be of importance, especially in the chamois population, where pestiviruses seem to circulate extensively.

The two sides of border disease in Pyrenean chamois (Rupicapra pyrenaica): silent persistence and population collapse.

Abstract In 2001, border disease virus (BDV) was identified as the cause of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica) in Spain. Since then, the disease has caused a dramatic decrease, and in some cases collapse, of chamois populations and has expanded to nearly the entire distribution area in the Pyrenees. Chamois BDV was characterized as BDV-4 genotype and experimental studies confirmed that it was the primary agent of the disease. The infection has become endemic in the Central and Eastern Pyrenees. However, while most Pyrenean chamois populations have been severely affected by the disease, others have not, despite the circulation of BDV in apparently healthy individuals, suggesting the existence of different viral strategies for persisting in the host population. Changes in the interplay of pathogen, host and environmental factors may lead to the formation of different disease patterns. A key factor influencing disease emergence may be pathogen invasiveness through viral mutation. Host factors, such as behavior, immunity at the population level and genetic variability, may also have driven different epidemiological scenarios. Climatic and other ecological factors may have favored secondary infections, such as pneumonia, that under particular circumstances have been major contributing factors in the high mortality observed in some areas.

Investigations of pestivirus infection in wild Caprinae in Europe.

PESTIVIRUSES (family Flaviviridae) are single-stranded, positive-sense RNA viruses that are traditionally classified in four species: bovine viral diarrhoea virus type 1 (BVDV-1) and type 2 (BVDV-2), which affect cattle, Border disease virus (BDV), which infects small ruminants and classical swine fever virus, which affects pigs. BVDV and BDV are not strictly host-specific and antibodies to these viruses have been reported in several species of wild ruminants (Vilcek and Nettleton 2006). Pestiviruses can cause a wide range of reproductive clinical manifestations in livestock, and recently, BDV infection has been associated with severe outbreaks of disease in the Pyrenean chamois ( Rupicapra pyrenaica ), leading to severe reductions in its populations (Marco and others 2007, 2009). The Alpine ibex ( Capra ibex ) and Iberian ibex ( Capra pyrenaica ) are mountain ruminants belonging to the subfamily Caprinae (family Bovidae). Although these species are not considered threatened by the International Union for Conservation of Nature, there is concern regarding their genetic diversity, the founder effect and minimum viable populations (Shackleton 1997). Therefore, information about reproductive disorders could be of importance for the conservation and management of these species. The objectives of the present study were to determine the prevalence of antibodies to pestiviruses and to investigate the …

Spatial and Temporal Phylogeny of Border Disease Virus in Pyrenean Chamois (Rupicapra p. pyrenaica).

Border disease virus (BDV) affects a wide range of ruminants worldwide, mainly domestic sheep and goat. Since 2001 several outbreaks of disease associated to BDV infection have been described in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in Spain, France and Andorra. In order to reconstruct the most probable places of origin and pathways of dispersion of BDV among Pyrenean chamois, a phylogenetic analysis of 95 BDV 5’untranslated sequences has been performed on chamois and domestic ungulates, including novel sequences and retrieved from public databases, using a Bayesian Markov Chain Monte Carlo method. Discrete and continuous space phylogeography have been applied on chamois sequences dataset, using centroid positions and latitude and longitude coordinates of the animals, respectively. The estimated mean evolutionary rate of BDV sequences was 2.9×10−3 subs/site/year (95% HPD: 1.5–4.6×10−3). All the Pyrenean chamois isolates clustered in a unique highly significant clade, that originated from BDV-4a ovine clade. The introduction from sheep (dated back to the early 90s) generated a founder effect on the chamois population and the most probable place of origin of Pyrenean chamois BDV was estimated at coordinates 42.42 N and 1.9 E. The pathways of virus dispersion showed two main routes: the first started on the early 90s of the past century with a westward direction and the second arise in Central Pyrenees. The virus spread westward for more than 125 km and southward for about 50km and the estimated epidemic diffusion rate was about 13.1 km/year (95% HPD 5.2–21.4 km/year). The strong spatial structure, with strains from a single locality segregating together in homogeneous groups, and the significant pathways of viral dispersion among the areas, allowed to reconstruct both events of infection in a single area and of migrations, occurring between neighboring areas.

Haematology and serum chemistry of Pyrenean chamois (Rupicapra pyrenaica) naturally infected with a border disease virus

In 2005 and 2006 an outbreak of disease associated with border disease virus (BDV) infection caused high mortality in the Pyrenean chamois (Rupicapra pyrenaica) in the Catalan Pyrenees (NE Spain). The aim of this study was to determine values for different haematological and serum biochemical analytes in 32 free-ranging Pyrenean chamois affected by the disease and to compare them with those obtained from healthy chamois. In the affected chamois red blood cell counts, haemoglobin concentrations, packed cell volumes, mean corpuscular volumes and lymphocyte counts were all lower, while the neutrophil and platelet counts were higher. Glucose, lactate, triglycerides, creatinine, total protein concentrations and alkaline phosphatase activity were also lower, in contrast to the concentrations of total bilirubin, urea and aspartate aminotransferase activity, which were higher. Most of the observed changes could be associated with cachexia and inflammation in the affected chamois. Lymphopenia could be directly related to the BDV, which would lead to immunosuppression and explain the high rate of secondary infection observed in these animals.